The development of the vertebrate nervous system is characterized by a series of complex events beginning with an apparently homogeneous neuroepithelium in the early embryo and leading to formation of diverse, highly ordered, and interconnected neural cell types in the adult. Considerable descriptive and experimental evidence has been amassed which points to the existence of limiting diffusible factors that are required for the targeting, survival, and proper synaptic arrangement of neurons (R. W. Oppenheim, In: Studies in Developmental Neurobiology. (Cowan, W. M. ed.), Oxford Univerity Press, pp. 74-133, 1981; W. D. Snider and E. M. Johnson, Ann. Neurol. 26:489-506 (1989)). Functional neuronal circuits are sculpted from an initially overabundant production of neurons during development. In the mid term embryo, a process of programmed cell death eliminates a major proportion of the neuron population, leaving behind the appropriate number of neurons required for innervation of target tissues (V. Hamburger and R. Levi-Montalcini, J. Exp. Zool. 111:457-502 (1949); Y.-A. Barde, Neuron 2:1525-1535 (1989)).
The discovery of Nerve Growth Factor (NGF) provided the first direct evidence for the existence of neurotrophic, polypeptide factors (R. Levi-Montalcini and V. Hamburger, J. Exp. Zool. 116:321-362 (1951); R. Levi-Montalcini and P. U. Angeletti, Physiol. Rev. 48:534-569(1968)). This has been followed by the more recent description of additional neurotrophic factors: BDNF, CTNF, and NT-3, (for review see W. D. Snider and E. M. Johnson, Ann. Neurol. 26:489-506 (1989); G. Barbin et al., J. Neurochem. 43:1468-1478 (1984); P. C. Maisonpierre et al., Science 247:1446-1451 (1990)). The physiological consequences elicited by NGF in vitro and in vivo have been at the center of research in neurobiology for several decades. Consequently, considerable information is now available about the cell types that respond to NGF in the peripheral and central nervous systems.
NGF is known to play a role in the targeting and survival of sympathetic and neural crest-derived sensory neurons as well as in selected populations of cholinergic neurons in the brain (L. A. Greene and E. M. Shooter, Annu. Rev. Neurosci. 3:353-402 (1980); H. Thoenen and Y.-A. Barde, Physiol. Rev. 60:1284-1335 (1980); H. Gnahn et al., Dev. Brain. Res. 9:45-52 (1983)). It appears that the NGF dependent cholinergic neurons in the basal forebrain correspond to the population of cells that undergo attrition of Alzheimer's disease (F. Hefti, Annals of neurology, 13:109-110 (1983); Hefti and Wemer, 1986; Johnson and Tanuchi, 1987; P. J. Whitehouse et al., Science 215:1237-1239 (1982)). In vivo studies, in which NGF was injected in the periphery of the mouse embryo trunk, result in enhanced survival of sensory ganglia that are normally targeted for cell death (V. Hamburger et al., J. Neurosci. 1:60-71 (1981); I. B. Black et al., In: Growth Factors and Development, Current Topics in Developmental Biology, vol. 24 (Nilsen-Hamilton, ed.), pp. 161-192 (1990)).
Exposure of embryos to NGF antibodies results in reduced survival of dorsal root ganglion neurons while injection of NGF antibodies into neonate mice has the principal effect of inhibiting the survival of sympathetic neurons (R. Levi-Montalcini and B. Booker, Proc. Natl. Acad. Sci. USA, 46:373-384 or 384-391 (1960); S. Cohen, Proc. Natl. Acad. Sic. USA, 46:302-311 (1960); E. M. Johnson et al., Science 210:916-918 (1980)).
In vitro, some tumor cell lines of neural origin respond to the presence of NGF by undergoing differentiation along neuronal pathways. PC12 cells, derived from a rat pheochromocytoma, are the best characterized of these cell lines and represent a widely accepted model for NGF-mediated response and for neuronal differentiation (L. A. Greene and A. S. Tischler, Proc. Natl. Acad. Sci. USA 73:2424-2428 (1976)).
Although much is understood about the biology of NGF outside the cell, the mechanisms by which NGF elicits neurotrophic effects within the cell have not been fully resolved. Interaction of NGF with a cell receptor is a requisite step in the transmission of neurotrophic signals within the cell (for review see M. V. Chao, In: Handbook of Experimental Pharmacology, vol. 95/II Peptide Growth Factors and Their Receptors II (Sporn, M. B. and Roberts, A. B. eds.), Springer-Verlag, Heidelberg, pp. 135-165 (1990)). A major advance in understanding NGF interactions with the cells was the identification and cloning of a 75kDa receptor (75kNGF-R) that binds NGF, and is present in NGF-responsive cells. The clones of the gene encoding 75kNG-R have been characterized from several species (M. V. Chao et al., Science 232:418-421 (1986); M. J. Radeke et al., Nature 325:593-597 (1987)). Unfortunately, the structural and biological properties of 75kNGF-R have provided limited clues about the nature of the NGF signal trandsuction pathway inside the cell. 75kNGF-R displays the binding properties of a low affinity NGF receptor (Kd.apprxeq.10.sup.-9 M) when expressed in exogenous cell lines and analysis of the intracellular domain has not revealed putative domains of catalytic action (M. V. Caho, In: Handbook of Experimental Pharmacology, Vol. 95/II Peptide Growth Factors and Their Receptors II (Sporn, M. B. and Roberts, A. B. eds.), Springer-Verlag, Heidelberg, pp. 135-165 (1990)).
The biological responsiveness to NGF, however, is widely held to depend upon interactions with a high affinity binding component implying that other receptor or receptor subunits may be involved in NGF responses. The search for potential second messengers that might transmit NGF signals in PC12 cells has led to recent evidence indicating that activation of tyrosine kinases may represent an early response to the presence of NGF (Maher 1988). These data implicate tyrosine kinases as candidates in the composition of a high affinity receptor.
The trk proto-oncogene encodes a tyrosine kinase (TK) receptor with a tightly regulated neural pattern of expression during murine development (D. Martin-Zanca et al., Genes Dev. 4:683-694 (1990); D. Martin-Zanca et al., In: The Avian Model in Developmental Biology: From Organism to Genes, Editions du CNRS - 1990, pp. 291-302 (1990)). In vivo, transcripts for this gene were observed only in neural crest-derived sensory neurons of the peripheral nervous system through E17 of mouse development. Several lines of evidence have led applicants to investigate the possible involvement of trk in pC12 cell NGF-mediated events.
The need exists in the field to determine whether trk proto-oncogene tyrosine kinase receptor is activated via direct interaction with NGF. The present invention provides a complex comprising NGF ligand and trk-proto-oncogene receptor. The direct binding of NGF to the trk receptor leads to tyrosine phosphorylation and tyrosine kinase activity in response to NGF exposure in trk expressing cells. Knowledge of the trk physiological receptor and cognate NGF complex may allow a detailed study of nerve growth and regeneration. Furthermore, the demonstration of NGF-trk receptor complexes demonstrates methods for identifying related tyrosine kinase receptors providing additional neurotropic-factor pairs.